The goal of this research is to elucidate the molecular mechanisms of cell division cycle control in eukaryotic cells. Our approach includes the enzymatic dissection of the cellular DNA replicating machinery and the identification and isolation of the proteins involved in the initiation of DNA replication in the cell cycle. For this purpose, the 2-micron DNA plasmid of yeast (Saccharomyces) is being used as a defined template for cell-free assays of DNA replication. This plasmid replicates under the same control as nuclear DNA in vivo. Once the initiation proteins are isolated, a study of the processes that control their appearance in the cell cycle will be undertaken, as a first step in the development of assay for molecules involved in this control. This approach should lead to a better understanding of the events proximal to initiation of DNA replication, and provide the basis for the study of earlier events in the cell cycle. The use of yeast in these studies possesses several advantages: large amounts of material can be prepared for biochemical analysis, conditional mutants that arrest at various specific points in the cell division cycle are available, and yeast cells are normal, untransformed growing cells. The basic similarity of cell cycle phenomena in all eukaryotes suggests that these studies with yeast may provide information applicable to cell cycle control processes important in diverse cellular responses, including neoplastic change. Thus, studies utilizing yeast can augment work in other laboratories utilizing transformed cells in tissue culture.